ES2382157T3 - Liquid composition of factor VII polypeptides - Google Patents

Abstract

The invention concerns a liquid aqueous composition comprising (i) a factor VII polypeptide, (ii) an agent suitable for keeping pH in the range of from about 4.0 to about 9.0; (iii) an agent selected from the group consisting of: a calcium salt, a magnesium salt, or a mixture thereof; wherein the concentration of (iii) is less than 15 mM; and (iv) An ionic strength modifying agent; wherein the ionic strength of the composition is at least 200 mM.

Description

Liquid composition of factor VII polypeptides.

FIELD OF THE INVENTION

[0001] The present invention relates to aqueous liquid compositions containing factor VII polypeptides, and to methods for the production and use of such compositions. More particularly, this invention relates to liquid compositions stabilized against physical and / or chemical degradation.

BACKGROUND OF THE INVENTION

[0002] A variety of factors involved in the blood coagulation process have been identified, including factor VII (FVII), a plasma glycoprotein. Hemostasis begins by forming a complex between the tissue factor (TF) that is exposed to the blood in circulation after a wound occurs in the wall of a vessel, and the FVIIa that is present in the circulation in a corresponding amount to about 1% of the total FVII protein mass. FVII exists in plasma primarily as a single stranded zymogen, which is divided by FXa into its activated double stranded form, FVIIa. The activated recombinant factor VIIa (rFVIIa) has been developed as a prohemostatic agent. The administration of rFVIIa offers a rapid and highly effective prohemostatic response in hemophilic subjects with bleeding that cannot be treated with other coagulation factor products due to antibody formation. Also bleeding in subjects with factor VII deficiency or subjects with a normal coagulation system but experiencing bleeding can be successfully treated with FVIIa.

[0003] It is desirable to have forms of administration of factor VIIa suitable for both storage and distribution. Ideally, the drug product is stored and administered as a liquid. Alternatively, the drug product is lyophilized, that is, freeze-dried, and then reconstituted by adding a suitable diluent just before patient use. Ideally, the drug product has sufficient stability for long-term storage, that is, more than six months.

[0004] The decision to keep the final drug product as a liquid or lyophilize it is usually based on the stability of the protein drug in such forms. Protein stability can be affected among other things by factors such as ionic strength, pH, temperature, repeated freeze / thaw cycles, and exposures to shear stresses. The active protein can be lost as a result of physical instabilities, including denaturation and aggregation (both the formation of soluble and insoluble aggregates), as well as chemical instabilities, including, for example, hydrolysis, deamidation and oxidation, to name just a few. For a general review of protein drug stability, see, for example, Manning, et al., Pharmaceutical Research 6: 903-918 (1989).

[0005] While the possible incidence of protein instabilities is greatly appreciated, it is impossible to predict the particular instability problems of a particular protein. Any of these instabilities can result in the formation of a protein by-product, or derivative, that has reduced activity, increased toxicity, and / or increased immunogenicity. In fact, protein precipitation can lead to thrombosis, a lack of homogeneity of the dosage form and quantity, as well as jammed syringes. In addition, post-translational modifications such as, for example, gamma carboxylation of certain glutamic acid residues in the N-terminal and the addition of carbohydrate side chains provide potential sites that may be susceptible to modification during storage. Also, specifically for factor VIIa, being a serine protease, fragmentation due to autocatalysis (enzymatic degradation) can occur. Thus, the safety and efficacy of any composition of a protein is directly related to its stability. Maintaining stability in a liquid form is generally different from a lyophilized form due to the considerably increased potential for molecular movement and, therefore, the increased probability of molecular interactions. Maintaining stability in a concentrated form is also different due to the propensity to aggregate formation in increased protein concentrations.

[0006] When developing a liquid composition, many factors are taken into account. In the short term, that is, less than six months, liquid stability generally depends on avoiding major structural changes, such as denaturation and aggregation. These processes are described in the literature for various proteins, and there are many examples of stabilizing agents. It is well known that an effective agent to stabilize one protein actually acts to destabilize another. Once the protein has been stabilized against major structural changes, developing a liquid composition for long-term stability (e.g., more than six months) depends on further stabilizing the protein from specific degradation types for that protein. . More specific types of degradation may include, for example, disulfide bond randomization, oxidation of certain residues, deamidation, cyclization. Although it is not always possible to specify the types of individual degradation, tests have been developed to control imperceptible changes to control the ability of specific excipients to stabilize only the protein of interest.

[0007] In addition to stability considerations, excipients are generally selected, which are approved by several worldwide regulatory medical agencies. It is desirable that the pH of the composition be in a physiologically suitable range in an injection / infusion, otherwise the patient may experience pain and discomfort.

[0008] For an overview of protein compositions, see, for example, Cleland et al.: The development of stable protein compositions: A closer look at protein aggregation, deamidation and oxidation, Critical Reviews in Therapeutic Drug Carrier Systems 1993, 10 (4): 307-377; and Wang et al., Parenteral compositions of proteins and peptides: Stability and stabilizers, Journal of Parenteral Science and Technology 1988 (supplement), 42 (2S).

[0009] Other publications of interest regarding protein stabilization are as follows. The U.S. document 20010031721 A1 (American Home Products) refers to highly concentrated, lyophilized, and liquid factor IX compositions. The U.S. document 5,770,700 (Genetics Institute) refers to liquid compositions of factor IX. WO 97/19687 (American Red Cross) refers to liquid plasma protein compositions, in particular factor VIII and factor IX. The U.S. document 4,297,344 discloses the stabilization of coagulation factors II and VIII, antithrombin III, and plasminogen against heat by adding selected amino acids such as glycine, alanine, hydroxyproline, glutamine, and aminobutyric acid, and a carbohydrate such as a monosaccharide, an oligosaccharide, or a sugar alcohol

[0010] US6310183 discloses a composition consisting of rFVIIa, sodium chloride (2.92 mg / ml), glycylglycine, polysorbate 80, calcium chloride (1.47 mg / ml) and mannitol at a pH of 5, 5.

[0011] Factor VIIa undergoes several degradation pathways, especially aggregation (dimerization), oxidation, and autolytic cleavage (clipping of the peptide skeleton). In addition, precipitation may occur. Many of these reactions can be significantly slowed by eliminating water from the protein. However, the development of an aqueous composition for factor VIIa has the advantages of eliminating reconstitution errors, thus increasing the accuracy of dosing, as well as simplifying the use of the product clinically, thus increasing the patient's adaptability. Ideally, factor VIIa compositions should be stable for more than 6 months over a wide range of protein concentrations. This allows flexibility in administration methods. Generally, more highly concentrated forms allow the administration of lower volumes, which is highly desirable from the point of view of patients. Liquid compositions can have many advantages over lyophilized products with respect to ease of administration and use.

[0012] Today, the only FVII polypeptide composition, obtained by recombination and commercially available, is a product of lyophilized FVIIa factor that is reconstituted before use; This contains a relatively low concentration of factor VIIa, for example, approximately 0.6 mg / ml. One vial (1.2 mg) of NovoSeven® (Novo Nordisk A / S, Denmark) contains 1.2 mg of recombinant human factor VIIa, 5.4 mg of NaCl, 2.94 mg of CaCl2, 2 H2O, 2, 64 mg of GlyGly, 0.14 mg of polysorbate 80, and 60.0 mg of mannitol; This is reconstituted at a pH of 5.5 by 2.0 ml of water for injection (WFI). When reconstituted, the protein solution is stable for use for 24 hours. Thus, no ready-to-use liquid or concentrated factor VII products are usually commercially available.

[0013] Accordingly, there is a need in the art for methods to improve the stability of factor VII polypeptides, including human factor VIIa (physical and / or chemical stability), increasing concentration, maintaining activity levels, and providing Liquid compositions suitable for storage. Thus, it is an objective of this invention to provide an aqueous composition of factor VII polypeptides that provides acceptable control of chemical and / or physical degradation products such as enzymatic degradation.

or autocatalysis products.

SUMMARY OF THE INVENTION

[0014] The present inventors have now discovered that factor VII or its analogues (i) ("factor VII polypeptides"), when formulated in the aqueous solution with an ionic strength of at least 200 mM are stable in the range of pH from about 4 to about 9.

[0015] Thus, in one aspect, the present invention provides an aqueous liquid composition comprising a factor VII polypeptide (i); a buffering agent (ii) suitable for maintaining the pH in the range of about 5.5 to about 9.0; an agent (iii) selected from the list of: a calcium salt, a magnesium salt, or a mixture thereof; where the concentration of (iii) is less than 15 mM; and an ionic strength modifying agent (iv); where the ionic strength of the composition is at least about 200 mM.

[0016] In a second aspect, the invention also provides a method for preparing a liquid aqueous composition of a factor VII polypeptide, comprising the step of providing the factor VII polypeptide in a solution comprising a buffering agent (ii) suitable for maintain the pH in the range of about 5.5 to about 9.0; an agent (iii) selected from the list of: a calcium salt, a magnesium salt, or a mixture thereof, where the concentration of (iii) is less than 15 mM; and an ionic strength modifying agent (iv); while ensuring that, in the final composition, the ionic strength of the composition is at least about 200 mM.

[0017] In a third aspect, the invention also relates to the use of a composition as defined above for use as a medicament.

[0018] In a fourth aspect, the invention also relates to the use of a composition as defined above for the preparation of a medicament for treating a factor VII sensitive syndrome.

[0019] In a fifth aspect, the invention relates to a method for reducing the degradation of factor VII in a liquid formulation, said method comprising the step of providing the factor VII polypeptide in a solution comprising a suitable buffering agent (ii) to maintain the pH in the range of about 5.5 to about 9.0; an agent (iii) selected from the list of: a calcium salt, a magnesium salt, or a mixture thereof; where the concentration of (iii) is less than 15 mM; and an ionic strength modification agent (iv); while ensuring that, in the final composition, the ionic strength is at least 200 mM.

[0020] In a seventh aspect, the invention relates to an airtight container, at least partially filled, containing a liquid aqueous pharmaceutical formulation as defined above, and optionally an inert gas, said container comprising (i) a part of wall and (ii) one or more closing means that do not constitute part of said wall part.

DETAILED DESCRIPTION OF THE INVENTION

[0021] As mentioned above, the present invention resides in the development of a new aqueous, liquid and stabilized pharmaceutical composition comprising a factor VII polypeptide. More specifically, the liquid aqueous pharmaceutical composition comprises a factor VII polypeptide (i), a buffer agent (ii) suitable for maintaining the pH in the range of about 5.5 to about 9.0, an agent (iii) selected from the list of: a calcium salt, a magnesium salt, or a mixture thereof; where the concentration of (iii) is less than 15 mM, and an ionic strength modifying agent (iv); where the ionic strength of the composition is at least about 200 mM.

[0022] The compositions according to the present invention are useful as stable compositions and preferably ready for use of factor VII polypeptides. In addition, it is believed that the specific principles, guidelines and embodiments given herein are equally applicable for the bulk storage of factor VII polypeptides, mutatis mutandis. The compositions are stable for at least six months, and preferably up to 36 months; when stored at temperatures in the range of 2 ° to 8 ° C. The compositions are chemically and / or physically stable, in particular chemically stable, when stored for at least 6 months at a temperature of 2 ° to 8 ° C.

[0023] The term "stable in storage" or the interchangeable "stable" encompasses a product that is stabilized with storage at temperatures between 2 ° C - 8 ° C and remains within the preselected product specifications for a period of time adequate, often several months.

[0024] The term "stable" is intended to cover a composition that, after storage for 6 months at 2 to 8 ° C, retains at least 50% of its initial biological activity as measured in a coagulation test of a stage, for example, essentially as described in test 4 of the present specification. Preferably, the stable composition retains at least 70%, such as 75% or 80% of its initial activity after storage for 6 months at a temperature of 2 to 8 ° C.

[0025] In addition, the term "stable" encompasses a composition that, after storage for at least 6 months at a temperature of 2 to 8 ° C, contains less (in%) of at least one of the following degradation products : (i) enzymatic degradation products, (ii) aggregates (dimers, oligomers, polymers), (iii) oxidized forms, or (iv) deamidated forms in relation to the amount of corresponding degradation product (s) contained in a solution of reconstituted NovoSeven® product that has been stored under similar conditions for a similar period of time.

[0026] The term "physically stable" of factor VII polypeptides refers to the formation of soluble and / or insoluble aggregates in the form of dimeric, oligomeric and polymeric forms of factor VII polypeptides as well as any structural deformation and denaturation of the molecule

[0027] The term "chemical stability" is intended to refer to the formation of any chemical change in factor VII polypeptides with storage in the solution under accelerated conditions. For example are hydrolysis, deamidation and oxidation, as well as enzymatic degradation, resulting in the formation of factor VII polypeptide fragments (such as heavy chain degradation). In particular, sulfur amino acids are prone to oxidation with the formation of the corresponding sulfoxides.

[0028] In interesting embodiments of the invention, suitable compositions have a limited increase in the content of enzymatic degradation forms with storage for at least 6 months at a temperature of 2-8 ° C. Interesting embodiments refer to compositions that are stable so that no more than about 25% w / w, such as 20%, 15%, 10%, or no more than about 5% of the initial polypeptide content Factor VII converts heavy chain fragments to enzymatic degradation with storage of said composition at a temperature of 2-8 ° C for 6 months.

[0029] The term "initial content" refers to the amount of factor VII polypeptides added to a composition in the preparation of the composition.

[0030] The compositions comprise polypeptides of factor VII (i), calcium and / or magnesium ions (iii), buffering agents (II), ionic strength modifying agents (iv) and, optionally, other excipients, which They also stabilize factor VII polypeptides, including detergents and tonicity modifiers. The concentration of factor VII polypeptides ranges from about 0.1 to about 15 mg / mL.

Factor VII polypeptide (i):

[0031] The terms "FVII" or "human factor VII" indicates human factor VII produced by methods that include natural source extraction and purification, and by recombinant cell culture systems. Its sequence and characteristics are set forth, for example, in U.S. Patent No. 4,784,950. The terms also encompass biologically active human factor VII equivalents, for example, which differ by one or more amino acid (s) in the total sequence. In addition, the terms used in this application are intended to cover the amino acid substitution, deletion and insertion variants of factor VII or posttranslational modifications. As used in this case "factor VII polypeptide" encompasses, without limitation, factor VII, as well as polypeptides related to factor VII. Factor VII related polypeptides include, without limitation, factor VII polypeptides that have been chemically modified in relation to human factor VII and / or contain one or more amino acid sequence alterations relative to human factor VII (i.e., variants of the factor VII), and / or contain truncated amino acid sequences relative to human factor VII (i.e., factor VII fragments). Such polypeptides related to factor VII may show different properties in relation to human factor VII, including stability, phospholipid binding, specific altered activity, and the like.

[0032] The term "factor VII" is intended to encompass factor VII polypeptides in their undivided (zymogenic) form, as well as those that have been proteolytically processed to produce their respective bioactive forms, which may be designated factor VIIa. Typically, factor VII is divided between residues 152 and 153 to produce factor VIIa. The term "Factor VII" also aims to encompass, without limitation, polypeptides with the amino acid sequence 1-406 of natural human factor VII (as disclosed in US Patent No. 4,784,950), as well as derived natural factor VII of other species, such as, for example, bovine, porcine, canine, murine, and salmon factor II. It also encompasses natural allelic variations of factor VII that may exist and occur from one individual to another. Also, the degree and location of glycosylation or other posttranslation modifications may vary depending on the host cells chosen and the nature of the cellular host environment.

[0033] As used herein "polypeptides related to factor VII" encompasses, without limitation, polypeptides that show substantially equal or improved biological activity in relation to natural human factor VII. These polypeptides include, without limitation, factor VII or factor VIIa that has been chemically modified and factor VII variants in which alterations of the specific amino acid sequence have been introduced, which modify or interrupt the bioactivity of the polypeptide.

[0034] It also encompasses polypeptides with a slightly modified amino acid sequence, for example, polypeptides with a modified N-terminal end that includes deletions or additions of N-terminal amino acids, and / or polypeptides that have been chemically modified in relation to factor VIIa human.

[0035] Factor VII related polypeptides, including factor VII variants, show substantially a bioactivity equal to or better than natural factor VII, including, without limitation, polypeptides with an amino acid sequence that differs from the natural factor VII sequence by insertion, deletion, or substitution of one or more amino acids.

[0036] Factor VII related polypeptides, including variants, which have substantially the same or improved biological activity relative to natural factor VIIa encompass those that show at least about 25%, such as, for example, at least about 50 %, at least about 75%, at least about 100%, at least about 110%, at least about 120%, or at least about 130% of the specific activity of natural factor VIIa that has been produced in the same cell type, when evaluated in one or more of a coagulation assay, proteolysis assay, or TF binding assay as described in the present specification.

[0037] In some embodiments, Factor VII polypeptides are Factor VII related polypeptides, in particular variants, where the relationship between the activity of said Factor VII polypeptide and the activity of native human Factor VIIa (natural FVIIa) is at least about 1.25 when analyzed in the "in vitro hydrolysis assay" (see "Assays", below); In other embodiments, the ratio is at least about 2.0; In other embodiments, the ratio is at least about 4.0. In some embodiments of the invention, factor VII polypeptides are equivalent to factor VII, in particular variants, where the ratio between the activity of said factor VII polypeptide and the activity of native human Factor VIIa (natural FVIIa) is at minus approximately 1.25 when evaluated in the "in vitro proteolysis assay" (see "Assays", below); In other embodiments, the ratio is at least about 2.0 in other embodiments, the ratio is at least about 4.0 in other embodiments, the ratio is at least about 8.0.

[0038] In some embodiments, the factor VII polypeptide is human factor VII, as disclosed, for example, in U.S. Patent No. 4,784,950 (natural factor VII). In some embodiments, the factor VII polypeptide is human factor VIIa. In a series of embodiments, factor VII polypeptides include polypeptides that show at least about 90%, such as, for example, at least about 100%, at least about 120%, at least about 140% , or at least about 160%, of the specific biological activity of human factor VIIa.

[0039] In some embodiments, factor VII polypeptides have an amino acid sequence that differs from the natural factor VII sequence by insertion, deletion, or substitution of one or more amino acids.

[0040] In a series of embodiments, factor VII polypeptides include polypeptides that show at least about 70%, preferably at least about 80%, more preferably at least about 90%, and more preferably at least about 95%, of identity with the natural factor VII sequence as disclosed in U.S. Patent No. 4,784,950. The homology / identity of the amino acid sequence is conveniently determined from aligned sequences, using a computer program suitable for sequence alignment, such as, for example, the ClustalW program, version 1.8, 1999 (Tompson et al., 1994 , Nucleic Acid Research, 22: 4673-4680).

[0041] Non-limiting examples of factor VII variants that have substantially the same or improved biological activity as natural factor VII include S52A-FVII, S60A-FVII (Lino et al., Arch. Biochem. Biophys. 352: 182192, 1998 ); L305V-FVII, L305V / M306D / D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII, V158T / M298Q-FVII, V158D / E296V / M298Q-FVII, K337A-FVII-V8Q-8 FVII, L305V / K337A-FVII, V158D / E296V / M298Q / L305V-FVII, V158D / E296V / M298Q / K337A-FVII, V158D / E296V / M298Q / L305V / K337A-FVII, K157-FVII, K157-FVII M298Q-FVII, V158D / E296V-FVII, V158D / M298K-FVII, and S336G-FVII; FVIIa variants that show increased proteolytic stability as disclosed in U.S. Patent No. 5,580,560; Factor VIIa that has been proteolytically divided between residues 290 and 291 or between residues 315 and 316 (Mollerup et al., Biotechnol. Bioeng. 48: 501-505, 1995); oxidized forms of factor VIIa (Kornfelt et al., Arch. Biochem. Biophys. 363: 43-54, 1999); FVII variants as disclosed in PCT / DK02 / 00189; FVII variants that show increased proteolytic stability as disclosed in WO 02/38162 (Scripps Research Institute); FVII variants with a modified Gla domain and showing an improved membrane junction as disclosed in WO 99/20767 (University of Minnesota); FVII variants as disclosed in WO 01/58935 (Maxigen ApS); FVII variants with increased biological activity compared to natural FVIIa as disclosed in WO 01/83725, WO 02/22776, WO 02/077218, PCT / DK02 / 00635, Danish patent application PA 2002 01423, Danish patent application PA 2001 01627; WO 02/38162 (Scripps Research Institute); and variants of FVIIa with an improved activity as disclosed in JP 2001061479 (Chemo-Sero-Therapeutic Res Inst.).

[0042] Examples of factor VII or polypeptides related to factor VII include, without limitation, natural factor VII, L305V-FVII, L305V / M306D / D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII, V158T / M298Q -FVII, V158D / E296V / M298Q-FVII, K337A-FVII, M298Q-FVII, V158D / M298Q-FVII, L305V / K337A-FVII, V158D / E296V / M298Q / L305V-FVII-V158 / 297 / F7 , V158D / E296V / M298Q / L305V / K337A-FVII, K157A-FVII, E296V-FVII, E296V / M298Q- FVII, V158D / E296V-FVII, V158D / M298K-FVII, and S336G-FVII, K3337-FVII L305V / V158D-FVII, L305V / E296V-FVII, L305V / M298Q-FVII, L305V / V158T-FVII, L305V / K337A / V158T-FVII, L305V / K337A / M298Q-FVII, L305-FVII, L305-FVII, L305-FVII, L305-FVII, L305-FVII, L305-FVII, L305-FVII, L305-FVII, L305-FVII, L305-FVII, L305-EVI6 K337A / V158D-FVII, L305V / V158D / M298Q-FVII, L305V / V158D / E296V-FVII, L305V / V158T / M298Q-FVII, L305V / V158T / E296V-FVII, L305V / E296V / E296V / 829 E296V / M298Q-FVII, L305V / V158T / E296V / M298Q-FVII, L305V / V158T / K337A / M298Q-FVII, L305V / V158T / E296V / K337A-FVII, L305V / V158D / K158D / K158D / V158D / V158D / K158D / V158D / V58D / V58D / V58D / V58D / KV / V58D / V58D / V58D / V158D / V158D / V158D / V158D / KV / V58D / V / V7 / V / V7 / V / V7 / V / V / V7 E296V / K337A-FVII, L305V / V158D / E296V / M298Q / K33 7A-FVII, L305V / V158T / E296V / M298Q / K337A-FVII, S314E / K316H-FVII, S314E / K316Q-FVII, S314E / L305V-FVII, S314E / K337A-FVII, S314E / S314E / S314E / S314-E FVII, S314E / M298Q-FVII, S314E / V158T-FVII, K316H / L305V-FVII, K316H / K337A-FVII, K316H / V158D-FVII, K316H / E296V-FVII, K316H / M298QV, F29I K316Q / L305V-FVII, K316Q / K337A-FVII, K316Q / V158D-FVII, K316Q / E296V-FVII, K316Q / M298Q-FVII, K316Q / V158T-FVII, S314E / L305V / K337V / K337- V157 / V157 / V3D / K337V FVII, S314E / L305V / E296V-FVII, S314E / L305V / M298Q-FVII, S314E / L305V / V158T-FVII, S314E / L305V / K337A / V158T-FVII, S314E / L305V / K33QV / K33 LV5V / K33 / V7 / K7 / L7 / V7L / K7 / S7V / K7V / K7V / K7V / K7 / L7 / V7L / K7 / L7 / V7 / L7 / V7 / L3 / V7 / L3 / V7 / L3 / V7 / L7 / V / L7 / V / L3 / V / L3 / V / L3 / R3 / L7 / R3 / L7 / V7 / R3 K337A / E296V-FVII, S314E / L305V / K337A / V158D-FVII, S314E / L305V / V158D / M298Q-FVII, S314E / L305V / V158D / E296V-FVII, S314E / L305V / S305V / V305V / S305V / V305V / L305V / V305V / L305V / V305V / V303V V158T / E296V-FVII, S314E / L305V / E296V / M298Q-FVII, S314E / L305V / V158D / E296V / M298Q-FVII, S314E / L305V / V158T / E296V / M298Q-FVII / S308 / F7I / K7Q / FV7 FVII, S314E / L305V / V158T / E296V / K337A-FVII, S314E / L305V / V158D / K337A / M298Q-FVII, S314E / L305V / V158D / E296V / K 337A-FVII, S314E / L305V / V158D / E296V / M298Q / K337A-FVII, S314E / L305V / V158T / E296V / M298Q / K337A-FVII, K316H / L305V / K337A-FVII, K316H / V330, K316H / V330, K316H / LV3, K316H / LV16, K316H / LV3, K316H / LV16, K316H / LV16, K316H / LV3, K316H / VVI3 L305V / E296V-FVII, K316H / L305V / M298Q-FVII, K316H / L305V / V158T-FVII, K316H / L305V / K337A / V158T-FVII, K316H / L305V / K337A / M298Q3 / K297Q / F29I FVII, K316H / L305V / K337A / V158D-FVII, K316H / L305V / V158D / M298Q-FVII, K316H / L305V / V158D / E296V-FVII, K316H / L305V / V158T / M298Q3 / V298T-V / E298Q3 / V68V-16 FVII, K316H / L305V / E296V / M298Q-FVII, K316H / L305V / V158D / E296V / M298Q-FVII, K316H / L305V / V158T / E296V / M298Q-FVII, K316H / L30TV / 830 / K316 / K316 / KV168 / KV163 L305V / V158T / E296V / K337A-FVII, K316H / L305V / V158D / K337A / M298Q-FVII, K316H / L305V / V158D / E296V / K337A FVII, K316H / L305V / V158H / K16V / V683 / KV6 / 313V / V158T / E296V / M298Q / K337A-FVII, K316Q / L305V / K337A-FVII, K316Q / L305V / V158D-FVII, K316Q / L305V / E296V-FVII, K316Q / L305V / M298Q-V / M298Q-V , K316Q / L305V / K337A / V158T-FVIII, K316Q / L305V / K337A / M298Q-FVII, K316Q / L305V / K337A / E296V-FVII, K316 Q / L305V / K337A / V158D-FVII, K316Q / L305V / V158D / M298Q-FVII, K316Q / L305V / V158D / E296V-FVII, K316Q / L305V / V158T / M298Q-FVII, K316Q / V530 / E3, K316Q / V3V / F3 K316Q / L305V / E296V / M298Q-FVII, K316Q / L305V / V158D / E296V / M298Q-FVII, K316Q / L305V / V158T / E296V / M298Q-FVII, K316Q / L305V / V158T / V158T / V158Q / V158T / V158T / V158T / V158T / V78 / V78 / V716 / K5 / Q7 / V716 / K5 / Q7 V158T / E296V / K337A-FVII, K316Q / L305V / V158D / K337A / M298Q-FVII, K316Q / L305V / V158D / E296V / K337A-FVII, K316Q / L305V / V158D / E296V / E296V / E296V / E296V / E296V / K295V / E296V / K293V / K716 / K5 V158T / E296V / M298Q / K337A-FVII, F374Y / K337A-FVII, F374Y / V158D-FVII, F374Y / E296V-FVII, F374Y / M298Q-FVII, F374Y / V158T-FVII, F374Y3, L374Y3, L374Y3, F374Y3 FVII, F374Y / L305V / K337A-FVII, F374Y / L305V / V158D-FVII, F374Y / L305V / E296V-FVII, F374Y / L305V / M298Q-FVII, F374Y / L305V / V158T-FVII-FVII-FVII-FVII-FVII F374Y / K337A / S314E-FVII, F374Y / K337A / V158T-FVII, F374Y / K337A / M298Q-FVII, F374Y / K337A / E296V-FVII, F374Y / K337A / V158D-FVII15, F37I-F37I / F373 V158D / M298Q-FVII, F374Y / V158D / E296V-FVII, F374Y / V158T / S314E-FVII, F374Y / V158T / M298Q-FVII, F374Y / V158T / E2 96V-FVII, F374Y / E296V / S314E-FVII, F374Y / S314E / M298Q-FVII, F374Y / E296V / M298Q-FVII, F374Y / L305V / K337A / V158D-FVII, F374Y / L305 / 637 / F74 / 6307 L305V / K337A / M298Q-FVII, F374Y / L305V / K337A / V158T-FVII, F374Y / L305V / K337A / S314E-FVII, F374Y / L305V / V158D / E296V-FVII, F374Y8V3 / F74 / L8V / F74V / FD4 / L3 L305V / V158D / S314E-FVII, F374Y / L305V / E296V / M298Q-FVII, F374Y / L305V / E296V / V158T-FVII, F374Y / L305V / E296V / S314E-FVII, F374Y / F84 / M8QV / F34 / M3 L305V / M298Q / S314E-FVII, F374Y / L305V / V158T / S314E-FVII, F374Y / K337A / S314E / V158T-FVII, F374Y / K337A / S314E / M298Q-FVII, F374Y3 K7E7 / K3E7 / K3E7 / K3E7 K337A / S314E / V158D-FVII, F374Y / K337A / V158T / M298Q-FVII, F374Y / K337A / V158T / E296V-FVII, F374Y / K337A / M298Q / E296V-FVII, F374Y7 K7Q / F74 / M3 K337A / E296V / V158D-FVII, F374Y / V158D / S314E / M298Q-FVII, F374Y / V158D / S314E / E296V-FVII, F374Y / V158D / M298Q / E296V-FVII, F374Y3148 / V3F / F4143 V158T / S314E / M298Q-FVII, F374Y / V158T / M298Q / E296V-FVII, F374Y / E296V / S314E / M298Q-FVII, F374Y / L305V / M298Q / K337A / S314E-FVII, F374Y / L305V / E296V / K337A / S314E-FVII, F374Y / E296V / M298Q / K337A / S314E-FVII, F374Y / L305V / E296V / M298Q / K337A / K337A / M337A / K337A-KI7V / K337A-M334A S314E-FVII, F374Y / V158D / E296V / M298Q / K337A-FVII, F374Y / V158D / E296V / M298Q / S314E-FVII, F374Y / L305V / V158D / K337A / S314E-FVIE / FVI7 / F77 / F77 / F7I / F77 / F7I / F77 / FY07 FVII, F374Y / V158D / E296V / K337A / S314E-FVII, F374Y / L305V / V158D / E296V / M298Q-FVII, F374Y / L305V / V158D / M298Q / K337A-FVII, F374Y6V / V5 / F74V6A / KV7V-6 F374Y / L305V / V158D / M298Q / S314E-FVII, F374Y / L305V / V158D / E296V / S314E-FVII, F374Y / V158T / E296V / M298Q / K337A-FVII, F374Y / V158T / F64E / V158T / F64E3 / V158T / F154T3 L305V / V158T / K337A / S314E-FVII, F374Y / V158T / M298Q / K337A / S314E-FVII, F374Y / V158T / E296V / K337A / S314E-FVII, F374Y / L305V / V158T / F6V / 815 / F6V / V68V / F6V / V68V / V6V / F6V / V68 / V68 / V68 / V68 / V68 / V68 / V68 / V68 / V68 / V68 / V68 / V6V / V6V / V6V / V6V3 V158T / M298Q / K337A-FVII, F374Y / L305V / V158T / E296V / K337A-FVII, F374Y / L305V / V158T / M298Q / S314E-FVII, F374Y / L305V / V158T / E296V / E296V / E296V / E296V / E296V / E296V / E296V / E296V / E296V / E296V / E296V / E296V / E296V / E296V / E296V / E296V / E296V / E296V / E296V / E296V / E296V / E296Y K337A / V158T / S314E-FVII, F374Y / V158D / E296V / M298Q / K337A / S314E-FVII, F374Y / L305V / V158D / E296V / M298Q / S314 E-FVII, F374Y / L305V / E296V / M298Q / V158T / S314E-FVII, F374Y / L305V / E296V / M298Q / K337A / V158T-FVII, F374Y / L305V / E296V / K337A / V158-VI3V / V158-V38 / V158-V303 M298Q / K337A / V158T / S314E-FVII, F374Y / L305V / V158D / E296V / M298Q / K337A-FVII, F374Y / L305V / V158D / E296V / K337A / S314E-FVII, F374Y8 / 314/837/293 / K3D / S7 FVII, F374Y / L305V / E296V / M298Q / K337A / V158T / S314E-FVII, F374Y / L305V / V158D / E296V / M298Q / K337A / S314E-FVII, S52A-factor VII, S60A VII factor; and P11Q / K33E-FVII, T106N-FVII, K143N / N145T-FVII, V253N-FVII, R290N / A292T-FVII, G291N-FVII, R315N / V317T-FVII, K143N / N145T / R315N / V315N / V315N / V315N / V315N / V315N / V315N / V315N FVII that has substitutions, additions or deletions in the amino acid sequence from 233Tr to 240Asn, FVII that has substitutions, additions or deletions in the amino acid sequence from 304Arg to 329Cys, and FVII that has substitutions, deletions, or additions in the sequence of amino acids Ile153-Arg223.

[0043] In different embodiments, the factor VII polypeptide is human factor VIIa; recombinant human factor VIIa; a factor VII related polypeptide; a sequence variant of factor VII; or a factor VII polypeptide where the activity of the factor VII polypeptide and the activity of the native human Factor VIIa (natural FVIIa) is at least about 1.25, preferably at least about 2.0 or 4.0, more preferably at least approximately 8.0, when evaluated in the "in vitro proteolysis assay" as described in the present specification. In one embodiment, the factor VII polypeptide has a different glycosylation than the natural human factor VII. In different embodiments, the factor VII polypeptide is present in a concentration of about 0.1 mg / ml to about 15 mg / ml; from about 0.5 mg / ml to about 10.0 mg / ml; from about 0.5 mg / ml to about 5.0 mg / ml; from about 0.6 mg / ml to about 4.0 mg / ml; from about 1.0 mg / ml to about 4.0 mg / ml; from about 0.1 mg / ml to about 5 mg / ml; from about 0.1 mg / ml to about 4.0 mg / ml; from about 0.1 mg / ml to about 2 mg / ml; or from about 0.1 mg / ml to about 1.5 mg / ml.

Buffering agent (ii):

[0044] To produce the liquid aqueous pharmaceutical composition useful for direct parenteral administration to a mammal such as a human, it is normally necessary for the pH value. of the composition is maintained within certain limits, such as from about 4.0 to about 9.0. To ensure a suitable pH value under the given conditions, the pharmaceutical composition also comprises a buffering agent (ii) suitable for maintaining the pH in the range of about 5.5 to about 9.0.

[0045] The term "buffering agent" encompasses those agents or combinations of agents that maintain the pH of the solution in an acceptable range of about 5.5 to about 9.0. These may include, but are not limited to, acids and salt of: MONTH, PIPES, ACES BES, TES, HEPES, TRIS, histidine (eg L-histidine), imidazole, glycine, glycylglycine, glycinamide, phosphoric acid ( eg sodium or potassium phosphate), acetic acid (eg, ammonium, sodium or calcium ketate), lactic acid, glutaric acid, citric acid (eg sodium or potassium citrate ), tartaric acid, malic acid, maleic acid, and succinic acid. It should be understood that the buffering agent may comprise a mixture of two or more components, where the mixture is capable of providing a pH value in the defined range. As an example it may be acetic acid and sodium acetate mentioned, or acetic acid and histidine, etc.

[0046] In one embodiment, the buffering agent (ii) is at least one component selected from the groups consisting of acids and salts of MES, PIPES, ACES, BES, TES, HEPES, TRIS, histidine (eg L-histidine), imidazole, glycine, glycylglycine, glycinamide, phosphoric acid (eg sodium or potassium phosphate), acetic acid (eg ammonium, sodium or calcium acetate), lactic acid, glutaric acid, citric acid (eg sodium or potassium citrate), tartaric acid, malic acid, maleic acid, and succinic acid.

[0047] In certain embodiments of the invention, the pharmaceutical composition comprises very small amounts of calcium; thus, it is possible to use a phosphoric acid based buffer system, that is, a phosphate buffer, without undesirable precipitation of calcium phosphates. Thus, in an interesting embodiment, the buffer is a phosphate buffer.

[0048] The concentration of the buffering agent is chosen to maintain the preferred pH of the solution. In several embodiments, the concentration of the buffering agent is 1-100 mM; 1-50 mM; 1-25 mM; or 2-20 mM.

[0049] In one embodiment, the pH of the composition is maintained from about 5.5 to about 9.0, such as from about 5.5 to about 7.5 from about 5.5 to about 7.0 of about 5.5 to about 6.5 from about 6.0 to about 7.5 from about 6.5 to about 7.5 or from about 6.0 to about 7.0 from about 6.4 to about 6.6 , or about 6.5, or about 5.5.

[0050] As used herein, the pH values specified as "approximately" are understood to be ± 0.1, for example approximately pH 8.0 includes pH 8.0 ± 0.1.

Agent with calcium and / or magnesium (iii):

[0051] The stable and liquid pharmaceutical composition comprises an agent (iii) selected from the list of a calcium salt, a magnesium salt, or a mixture thereof. The concentration of agent (iii) is less than 15mM. In various embodiments, agent (iii) is present in a concentration of at least about 0.1 µM; at least about 0.5 μM; at least about 1 μM; at least about 5 μM; at least about 10 μM; at least about 50 μM; at least about 100 μM; at least about 1 mM; at least about 2 mM; at least about 5 mM; at least about 10mM.

[0052] In several embodiments, the molar ratio between non-complex and / or magnesium ions (Ca2 + / Mg2 +) and FVII polypeptide is: 0.001-750; 0.001-250; 0.001-100; 0.001-10; 0.001-1.0; 0.001-0.5; 0.5-750; 0.5-250; 0.5-100; 0.510; 0.5-1.0; 0.001-0.4999; 0.005-0.050.

[0053] When used herein, the term "the concentration of non-complex calcium and / or magnesium ions" is intended to refer to the difference between the total concentration of calcium and / or magnesium ions and the concentration of calcium and / or magnesium linked to calcium / magnesium chelating agents. In this regard, the factor VII polypeptide is not considered a "calcium / magnesium chelating agent" although calcium and / or magnesium is expected to bind with, or be associated with, the factor VII polypeptide under certain conditions.

[0054] In one embodiment of the present invention, the molar ratio of non-complex calcium and / or magnesium ions (Ca2 + / Mg2 +) to the factor VII polypeptide is less than 0.5, for example, in the range of 0.001-0.499, such as 0.005-0.050, or in the range of 0.000-0.499, such as in the range of 0.000-0.050, or about 0.000. In an embodiment of the present invention, the molar ratio of non-complex calcium (Ca2 +) to the factor VII polypeptide is less than 0.5, for example in the range of 0.001-0.499, such as 0.005-0.050, or in the range of 0.000-0.499, such as in the range of 0.000-0.050, or about 0.000.

[0055] In another embodiment, the molar ratio of non-complex calcium and / or magnesium ions to the factor VII polypeptide is greater than 0.5. In another embodiment, the molar ratio of non-complex calcium ions to the factor VII polypeptide is greater than 0.5.

[0056] To obtain the low relative relationship between calcium and / or magnesium ions (Ca2 +) and the factor VII polypeptide, it may be necessary or desirable to remove excess calcium and / or magnesium ions, for example, by contact of the composition with an ion exchange material under conditions suitable for the removal of Ca2 + and / or Mg2 +, or adding a calcium / magnesium chelating agent to bind the excess (complex) ions of calcium and / or magnesium. This is particularly relevant when the ratio between calcium and / or magnesium ions and the factor VII polypeptide in a one-step solution of the process that precedes the formulation step exceeds the limit set above. Examples of "calcium / magnesium chelating agents" include EDTA, citric acid, NTA, DTPA, tartaric acid, lactic acid, malic acid, succinic acid, HIMDA, ADA and similar compounds.

[0057] In one embodiment, the calcium salt is selected from the list of: calcium chloride, calcium acetate, calcium gluconate, calcium levulate, or a mixture thereof.

[0058] In one embodiment, the magnesium salt is selected from the list of: magnesium chloride, magnesium acetate, magnesium sulfate, magnesium gluconate, magnesium levulate, magnesium salts of strong acids, or a mixture thereof .

[0059] In a preferred embodiment, agent (iii) comprises Ca2 +.

[0060] In preferred embodiments, agent (iii) is selected from the list of: calcium chloride, calcium acetate, magnesium chloride, magnesium acetate, magnesium sulfate, or a mixture thereof; and the ionic strength modifying agent (iv) is sodium chloride or a mixture of sodium chloride and at least one additional ionic strength modifying agent.

Ionic force modifying agent (iv):

[0061] As used herein, the term "ionic strength modifying agent" includes agents, which contribute to the ionic strength of the solution. Agents include, but are not limited to, neutral salts, for example, sodium chloride or potassium chloride; amino acids; small peptides (eg, having 2 to 5 amino acid residues such as, for example, glycylglycine), or a mixture of at least two of said modifying agents. The "ionic strength modifying agent" may also be a mixture of two or more such agents that in combination are capable of modifying the ionic strength at a level as defined in the present invention. Preferred agents are sodium chloride or a mixture of sodium chloride with one or more small peptides or amino acids. The ionic strength modifying agent (s) is present in a concentration sufficient to raise the ionic strength of the solution to at least 200 mM (calculated based on millimolar concentrations of agents). The ionic strength modifying agents may, for example, without limitation, each be present in a concentration of at least about 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 200 mM, 400 mM, 800 mM, 1000 mM, 1200 mM, 1500 mM, 1800 mM, 2000 mM, or at least 2200 mM.

[0062] The term "ionic force" is the ionic strength of the solution (μ) which, in the present context, is defined

TIFF (uncompressed) decompress

are needed to see this picture.

using the equation: μ = QuickTime ™ and a L Ci (Zi2), where μ is the ionic force, Ci is the molar concentration of an ion, and Zi is the charge (+ or -) of that ion (see, for example , Solomon, Journal of Chemical Education, 78 (12): 1691-92, 2001; James Fritz and George Schenk: Quantitative Analytical Chemistry, 1979).

[0063] In various embodiments of the invention, the ionic strength of the composition is at least 200 mM, such as at least 250 mM, at least 300 mM, at least 400 mM, at least 500 mM, at least 650 mM , at least 800 mM, at least 1000 mM, at least 1200 mM, at least 1600 mM, at least 2000 mM, at least 2400 mM, at least 2800 mM, or at least 3200 mM (ionic strength calculations based on concentrations millimolar (mM) components).

[0064] In different embodiments, the ionic strength modifying agent (iv) is selected from the list of: a neutral salt, for example, sodium chloride; an amino acid; or a small peptide, or a mixture of at least two of said modifying agents. In one embodiment, the ionic strength modifying agent comprises sodium chloride. In another embodiment the ionic strength modifying agent is sodium chloride.

[0065] By "neutral salt" is meant a salt that is neither an acid nor a base when dissolved in the aqueous solution. Examples include sodium chloride or potassium chloride.

[0066] In important embodiments, the pharmaceutical composition is adapted for subcutaneous, intravenous or intramuscular injection according to methods known in the art. The possibly high concentration of salts may be disadvantageous for certain groups of patients. Therefore, the present invention also provides a method before use to reduce the salt concentration in a liquid aqueous pharmaceutical composition, wherein said method comprises the step of contacting the liquid aqueous pharmaceutical composition defined herein with an exchange material of ions, a material suitable for desalination, and / or the dilution step of the composition.

[0067] The ion exchange material is preferably contained in a sterile container, for example, in a glass or plastic cartridge.

[0068] The liquid aqueous pharmaceutical composition is expected to be in contact with the ion exchange material, for example, by passing through a cartridge containing the ion exchange material, immediately before use. In a particular embodiment, the cartridge is intended to be an integral part of a syringe assembly.

Other ingredients:

Tonicity Modifier:

[0069] As used in this case, the term "tonicity modifier" includes agents, which contribute to the osmolality of the solution. Tonicity modifiers include, but are not limited to, amino acids; small peptides (eg, having 2 to 5 amino acid residues); neutral salts; mono- or disaccharides; polysaccharides; polyols, or a mixture of at least two of said modifiers. Examples of tonicity modifiers include, but are not limited to, sodium chloride, potassium chloride, sodium citrate, sucrose, glucose, glycylglycine, trehalose, and mannitol. Typically, modifiers are present in a concentration of about 1 to about 500 mM; from about 1 to about 300 mM; from about 10 to about 200 mM; or from about 20 to about 150 mM, depending on the other ingredients present. Neutral salts such as, for example, sodium chloride or potassium chloride can be used.

[0070] In one embodiment, the pharmaceutical composition comprises a tonicity modifying agent (v). In one embodiment, the tonicity modifying agent (v) is selected from the list of: a neutral salt; a mono-, di- or polysaccharide; a sugar alcohol; an amino acid; or a small peptide, or a mixture of at least two of said modifying agents.

Surfactant

[0071] Optionally, the compositions may also contain a surfactant or detergent (vi). "Surfactants" or "detergents" generally include those agents that protect the protein from induced air / solution interface stresses and induced solution / surface stresses (eg, resulting in protein aggregation). The detergent is preferably a non-ionic detergent including, without limitation, polysorbates (eg Tween®), such as polysorbate 20 or 80 polyoxyethylene alkyl ethers or poloxamers, such as poloxamer 188 or 407, (eg, Pluronic® polyols) and other block polymers of ethylene / polypropylene, or polyethylene glycol (PEG) such as PEG8000. The amount of surfactant present ranges from about 0.005 to about 2.0%.

[0072] In several embodiments, the nonionic surfactant is a polysorbate or a poloxamer or a polyoxyethylene alkyl ether; preferably poloxamer 188 or poloxamer 407, or polysorbate 20 or polysorbate 80, or polyoxy lauryl ether 23.

Antioxidants

[0073] Optionally, the composition may include an antioxidant (vii). Antioxidants include, but are not limited to, ascorbic acid, cysteine, homocysteine, cystine, cystathionine, methionine, glutathione, and other peptides with cysteine or methionine, in particular peptides with 2 to 5 amino acid residues where at least one of the residues is a methionine or cysteine residue; Methionine is preferred, in particular L-methionine. The antioxidant is included in a concentration of 0.1 to 5 mg / ml, such as 0.1 to 4; 0.1 to 2; or 0.5 to 2 mg / ml.

[0074] In one embodiment the antioxidant (vii) is selected from the list of: L- or D-methionine, a methionine analogue, a peptide containing methionine, a homologue of methionine, ascorbic acid, cysteine, homocysteine, glutathione, cystine, and cystathionine. In one embodiment, the antioxidant is L-methionine.

Preservative:

[0075] A preservative (viii) can also be included in the composition to retard microbial growth and thus allow "multiple use" packaging of FVII polypeptides. Preservatives include phenol, benzyl alcohol, ortho-cresol, meta-cresol, para-cresol, methyl paraben, propyl paraben, benzalkonium chloride, and benzethonium chloride. The preservative is normally included in a concentration of 0.1 to 20 mg / ml depending on the pH range and the type of preservative.

[0076] Optionally, the composition may also include an agent capable of inhibiting deamidation and / or isomerization.

[0077] As used herein, it is understood that the specific amounts are ± about 10%, for example, about 50 mM includes 50 mM ± 5 mM; for example, 4% includes 4% ± 0.4%, etc.

[0078] The percentages are (weight / weight) both when referring to solids dissolved in the solution and liquids mixed in solutions. For example, for Tween, it is the weight of 100% material / weight of the solution.

[0079] The term "isotonic" means "isotonic with serum", that is, at approximately 300 ± 50 millionmol / kg. The tonicity aims to be a measure of osmolality of the solution before administration. The term "hypertonic" is intended to designate osmolality levels above the physiological level of the serum, such as levels above 300 ± 50 milliosmol / kg.

[0080] In one embodiment, the composition is isotonic; In another, it is hypertonic. In one embodiment, the composition is formulated for pharmaceutical administration. In one embodiment, the composition is stable for storage for at least 6 months at 2-8 ° C.

Methods of use:

[0081] The compositions or preparations of the present invention can be used to treat any factor VII sensitive syndrome, such as, for example, blood disorders, including, without limitation, those caused by deficiencies in coagulation factors (p eg, hemophilia A and B or coagulation factor deficiency XI or VII); for thrombocytopenia or von Willebrand's disease, or for clotting factor inhibitors, or excessive bleeding from any cause. The preparations may also be administered to patients in combination with surgery or other trauma or to patients receiving anticoagulant therapy.

[0082] The term "pharmaceutically effective amount" or "effective amount" is the effective dose to be determined by a qualified professional, who can assess the dosages to achieve the desired response. Factors to consider in the doses include potency, bioavailability, desired pharmacokinetic / pharmacodynamic profiles, condition of treatment, factors related to the patient (eg weight, health, age, etc.), presence of co-administered medications (for example, anticoagulants), time of administration, or other factors known to a medical professional.

[0083] The term "treatment" is defined as the supervision and care of a subject, for example a mammal, in particular a human, for the purpose of combating the disease, condition, or disorder and includes the administration of a factor polypeptide. VII to prevent the onset of symptoms or complications, or relieve symptoms or complications, or eliminate disease, condition, or disorder. Pharmaceutical compositions according to the present invention with a factor VII polypeptide can be administered parenterally to subjects in need of such treatment. Parenteral administration can be performed by subcutaneous, intramuscular or intravenous injection by a syringe, optionally a pen syringe. Alternatively, parenteral administration can be performed by an infusion pump.

[0084] The concentration of factor VIIa is conveniently expressed as mg / mL or as IU / mL, with 1 mg normally representing 43000-56000 IU or more.

[0085] In different embodiments, the syndrome is selected from the group consisting of hemophilia A, hemophilia B, factor XI deficiency, factor VII deficiency, thrombocytopenia, von Willebrand disease, presence of a clotting factor inhibitor, surgery, intracerebral hemorrhage, trauma, stem cell transplantation, liver disease, dilution coagulopathy, upper digestive hemorrhages, and anticoagulant therapy.

General Methods

Suitable assays to determine the biological activity of factor VII polypeptides:

[0086] The biological activity of factor VIIa in blood coagulation derives from its ability to (i) bind to tissue factor (TF) and (ii) catalyze proteolytic cleavage of factor IX or factor X to produce factor IX or X activated (factor IXa or Xa, respectively).

[0087] For purposes of the invention, the biological activity of factor VII polypeptides ("factor VII biological activity") can be quantified by measuring the ability of a preparation to stimulate blood coagulation using factor VII deficient plasma and thromboplastin, as described, for example, in U.S. Patent No. 5,997,864 or WO 92/15686. In this assay, the biological activity is expressed as the reduction in coagulation time in relation to a control sample and is converted to "factor VII units" by comparison with a combined human serum standard containing 1 unit / ml of factor VII activity. Alternatively, the biological activity of factor VIIa can be quantified by

-

 measurement of the ability of factor VIIa or a polypeptide related to factor VII to produce activated Factor X (Factor Xa) in a system comprising TF introduced into a lipid membrane and factor X. (Persson et al., J. Biol. Chem 272: 19919-19924, 1997);

-

 measurement of Factor X hydrolysis in an aqueous system ("In vitro proteolysis test", see below);

-

 measurement of the physical binding of factor VIIa or a factor VII related polypeptide to TF using an instrument based on surface plasmon resonance (Persson, FEBS Letts. 413: 359-363, 1997);

-

 measurement of the hydrolysis of a synthetic substrate by Factor VIIa and / or a polypeptide related to factor VII ("In vitro hydrolysis assay", see below);

-

 measurement of thrombin generation in an in vitro system independent of TF.

[0088] Factor VII polypeptides useful according to the present invention can be selected by suitable assays that can be performed as simple preliminary tests in vitro. Thus, the present specification discloses a simple test (called "In vitro hydrolysis assay") for the activity of polypeptides of factor VII.

In vitro hydrolysis test (Test 1)

[0089] The native (natural) factor VIIa and the factor VII polypeptide (both hereafter referred to as "factor VIIa") can be evaluated for specific activities. These can also be evaluated in parallel to directly compare their specific activities. The test is carried out on a microtiter plate (MaxiSorp, Nunc, Denmark). The chromogenic substrate D-Ile-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden), final concentration 1 mM, is added to factor VIIa (final concentration of 100 nM) in 50 mM Hepes, pH 7.4 , with 0.1 M NaCl, 5 mM CaCl2 and 1 mg / ml bovine serum albumin. The absorbance at 405 nm is measured continuously on a SpectraMax ™ 340 plate reader (Molecular Devices, USA). The absorbance developed during a 20 minute incubation, after subtraction of the absorbance in a blank well without any enzyme, is used to calculate the relationship between the activities of the polypeptide of factor VII and the natural factor VIIa:

Ratio = (A405 nm of factor VII polypeptide) / (A405 nm of natural factor VIIa).

[0090] Based on this, factor VII polypeptides with a lower, comparable or superior activity than native factor VIIa can be identified, such as, for example, factor VII polypeptides where the relationship between factor VII polypeptide activity and the activity of native factor VII (natural FVII) is approximately, versus above 1.0.

[0091] The activity of factor VII polypeptides can also be measured using a physiological substrate such as factor X ("In vitro proteolysis assay"), suitably at a concentration of 100-1000 nM, where Factor Xa generated is measured after the addition of a suitable chromogenic substrate (for example, S-2765). In addition, the activity test can be performed at a physiological temperature.

In vitro proteolysis test (Test 2)

[0092] Native (natural) Factor VIIa and Factor VII polypeptide (both hereafter referred to as "Factor VIIa") are evaluated in parallel to directly compare their specific activities. The test is carried out on a microtiter plate (MaxiSorp, Nunc, Denmark). Factor VIIa (10 nM) and Factor X (0.8 microM) in 100 microL 50 mM Hepes, pH 7.4, with 0.1 M NaCl, 5 mM CaCl2 and 1 mg / ml serum albumin bovine, incubate for 15 min. The cleavage of Factor X is then stopped by the addition of 50 microL 50 mM Hepes, pH 7.4, with 0.1 M NaCl, 20 mM EDTA and 1 mg / ml bovine serum albumin. The amount of Factor Xa generated is measured by the addition of the chromogenic substrate z-D-Arg-Gly-Arg-P-nitroanilide (S-2765, Chromogenix, Sweden), 0.5 mM final concentration. The absorbance at 405 nm is measured continuously on a SpectraMax ™ 340 plate reader (Molecular Devices, USA). The absorbance developed over 10 minutes, after subtraction of the absorbance in a blank well without any FVIIa, is used to calculate the relationship between proteolytic activities of the factor VII polypeptide and the natural factor VIIa:

Ratio = (A405 nm polypeptide factor VII) / (A405 natural factor VIIa).

[0093] Based on this, the factor VII polypeptide with a lower, comparable or superior activity than the native factor VIIa can be identified, such as, for example, factor VII polypeptides where the relationship between the factor VII polypeptide activity and the activity of native factor VII (natural FVII) is approximately, versus above 1.0.

[0094] The ability of factor VIIa or factor VII polypeptides to generate thrombin can also be measured in an assay (Test 3) comprising all relevant coagulation factors and inhibitors in physiological concentrations (minus factor VIII when simulating the conditions of hemophilia A) and activated platelets (as described on page 543 in Monroe et al. (1997) Brit. J. Haematol. 99, 542-547, which is incorporated herein by reference).

[0095] The activity of factor VII polypeptides can also be measured using a one-stage coagulation assay (Test 4) essentially as described in WO 92/15686 or US 5,997,864. Briefly, the sample to be evaluated is diluted in 50 mM Tris (pH 7.5), 0.1% BSA and 100 µl is incubated with 100 µl of factor VII deficient plasma and 200 µl of thromboplastin C with 10 mM Ca2 +. Coagulation times are measured and compared with a standard curve using a reference standard or a mixture of normal human plasma citrated in serial dilution.

Preparation and purification of factor VII polypeptides:

[0096] Purified human factor VIIa suitable for use in the present invention is preferably obtained by recombinant DNA technology, for example as described in Hagen et al., Proc.Natl.Acad.Sci. USA 83: 2412-2416, 1986, or as described in European Patent No. 200,421 (ZymoGenetics, Inc.). Factor VII can also be produced by the methods described by Broze and Majerus, J.Biol.Chem. 255 (4): 1242-1247, 1980 and Hedner and Kisiel, J.Clin.Invest. 71: 1836-1841, 1983. These methods produce factor VII without detectable amounts of other blood coagulation factors. Another preparation of purified factor VII can be obtained by including an additional gel filtration as the final purification step. Factor VII is then converted into factor VIIa activated by known means, for example, by different plasma proteins, such as factor XIIa, IXa or Xa. Alternatively, as described by Bjoern et al. (Research Disclosure, September 269, 1986, pp. 564-565), factor VII can be activated by passing it through an ion exchange chromatography column, such as Mono Q® (Pharmacia fine Chemicals) or similar, or by solution self-activation.

[0097] Factor VII related polypeptides can be produced by modifying natural factor VII or by recombinant technology. Factor VII related polypeptides with altered amino acid sequence compared to natural factor VII can be produced by modifying the nucleic acid sequence encoding natural factor VII either by altering the amino acid codons or by removing some of the codons from amino acids in the nucleic acid encoding natural factor VII by known means, for example, by site specific mutagenesis.

[0098] It will be apparent to those skilled in the art that substitutions can be made outside the regions critical for the function of the factor VIIa molecule and still result in an active polypeptide. Essential amino acid residues for factor VII polypeptide activity, and therefore preferably not subject to substitution, can be identified according to methods known in the art, such as directed mutagenesis or alanine scanning mutagenesis (see, for example, Cunningham and Wells, 1989, Science 244: 1081-1085). In this latter technique, mutations are introduced into each positively charged residue in the molecule, and the resulting mutant molecules are evaluated for coagulant activity, respectively cross-linking to identify amino acid residues that are critical to the activity of the molecule. Enzyme-substrate interaction sites can also be determined by three-dimensional structure analysis as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (see, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

[0099] The introduction of a mutation in the nucleic acid sequence to exchange a nucleotide for another nucleotide can be performed by directed mutagenesis using any of the methods known in the art. Particularly useful is the procedure that uses a double-stranded supercoiled DNA vector with an insert of interest and two synthetic primers with the desired mutation. Oligonucleotide primers, each complementary to opposite strands of the vector, are extended during cyclic temperature variation by Pfu DNA polymerase. With the incorporation of the primers, a mutated plasmid with stepped cuts is generated. After the temperature cycle, the product is treated with DpnI, which is specific for methylated and hemimethylated DNA to digest the parental DNA template and to select the synthesized DNA that contains the mutation. Other methods known in the art to create, identify and isolate variants can be used, such as, for example, genetic transposition or phage display techniques. The separation of polypeptides from their cell of origin can be achieved by any method known in the art, including, without limitation, the removal of cell culture medium with the desired product from an adherent cell culture; centrifugation or filtration to remove non-adherent cells; and the like

[0100] Optionally, factor VII polypeptides can be further purified. Purification can be achieved using any method known in the art, including, without limitation, affinity chromatography, such as, for example, on a column of anti-factor VII antibodies (see, for example, Wakabaiashi et al., J. Biol. Chem. 261: 11097, 1986; and Thim et al., Biochem. 27: 7785, 1988); hydrophobic interaction chromatography; ion exchange chromatography; size exclusion chromatography; electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction and the like. See, generally, Scopes, Protein Purification, Springer-verlag, New York, 1982 and Protein Purification, JC Janson and Lars Ryden, editors, VCH Publishers, New York, 1989. Following the purification, the preparation preferably contains less than about 10% by weight, more preferably less than about 5% and of the more preferably less than about 1%, of non-factor VII polypeptides derived from the host cell.

[0101] Factor VII polypeptides can be activated by proteolytic cleavage, using factor XIIa or other proteases with trypsin type specificity, such as, for example, factor IXa; Kalikrein, factor Xa, and thrombin. See, for example, Osterud et al., Biochem. 11: 2853 (1972); Thomas, U.S. Patent No. 4,456,591; and Hedner et al., J. Clin. Invest. 71: 1836 (1983). Alternatively, factor VII polypeptides can be activated by passing them through an ion exchange chromatography column, such as Mono Q® (Pharmacia) or the like, or by self-activation in the solution. The resulting activated Factor VII polypeptide can then be formulated and administered as described in the present application.

DESCRIPTION OF THE FIGURES

[0102] Figure 1 shows contents of FVII aggregates and FVII fragments after 3 months of storage at 2-8 ° C.

[0103] The following examples illustrate the practice of the invention. These examples are for illustrative purposes only and are not intended in any way to limit the scope of the claimed invention.

EXAMPLES

Example 1

Analytical methods used in the determination of stability indication parameters:

A. Determination of oxidized forms by reverse phase HPLC (RP-HPLC):

[0104] HPLC column: 4.5x250 mm column filled with butyl bonded silica with a particle size of 5μm and pore size of 300Å. Column temperature: 70 ° C. Eluent A: 99.9% V / V water and 0.1% V / V trifluoroacetic acid. Eluent B: 80% V / V acetonitrile. 0.09% V / V trifluoroacetic acid and 19.91% V / V water. The column was eluted with a linear gradient from X% B to (X + 13)% B in 30 minutes. Flow rate: 1.0 ml / min. Detection: 214 nm.

[0105] The oxidized forms are methionine sulfoxides of factor VII polypeptides. For example, the two main derivatives of FVII are Met (O) 298 FVII and Met (O) 306 FVII.

[0106] The content of oxidized forms is expressed as the percentage of the initial amount of factor VII in the composition in the preparation that is recovered as oxidized forms of factor VII.

[0107] B. Determination of aggregates of factor VII polypeptides by high performance gel permeation chromatography (GP-HPLC).

[0108] GP-HPLC was carried out on a Waters Protein Pak 300 SW 7.5x300 mm column using 0.2 M ammonium sulfate pH 7.0 with 5% isopropanol as the mobile phase. Flow rate: 0.5 ml / min and detection: 215 nm.

[0109] The content of aggregates is expressed as the percentage of the initial amount of factor VII in the composition in the preparation that is recovered as dimeric, oligomeric and polymeric forms of factor VII.

Example 2

5 Composition preparation

[0110] In general, aqueous samples of the FVIIa composition for analysis in these experimental examples were prepared from a bulk solution purified by buffer exchange on a gel filtration column. The additives of the composition were well contained in the elution buffer in its final proportions or added to the eluate. The resulting solution was sterile filtered using a sterile membrane filter (0.2 micron pore size or equivalent) and introduced into sterile glass vials, with a cap and sealed with butyl rubber stoppers and aluminum flip-off caps.

15 Example 3 (comparative example)

Effect of pH on chemical / physical stability

[0111] (I) Vials of the aqueous composition of rFVIIa with 1.4 mg of rFVIIa / ml, 50 mM of sodium chloride, 10 mM of calcium chloride and a mixture of 10 mM of glycylglycine, acetate and histidine adjusted to pH 3; 3.5; 4.0; 4.5; 5.5; 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; and 9.0 were incubated at either a temperature of 2-8 ° C, or at elevated storage temperatures of 30 ° C, and then removed at various time points and evaluated for changes in pH and chemical stability was determined by RP-HPLC and GP-HPLC.

[0112] After storage at 2-8 ° C for a period of up to three months, the aqueous compositions showed negligible changes in pH. The non-denaturing HPLC by size exclusion that was performed on samples stored for a period of up to three months at 2-8 ° C showed no significant aggregation of the drug product at pH 5.5 values (Figure 1). The RP-HPLC performed on these samples showed no significant increase in fragmentation or oxidation of the protein in the pH range of 4.5-5.5.

[0113] Figure 1 shows data after 3 months of storage at 2-8 ° C. The initial content of aggregates is approximately 0.5% and the initial content of fragments is approximately 9%.

[0114] (II) Vials of the aqueous composition of rFVIIa containing 1.4 mg of rFVIIa / ml, 200 mM sodium chloride,

10 mM calcium chloride and a mixture of 10 mM glycylglycine, acetate and histidine adjusted to pH 3; 3.5; 4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; and 9.0 are incubated either at a temperature of 2-8 ° C, or at elevated storage temperatures of 30 ° C, and then removed at various points in time and evaluated for changes in pH; Chemical stability is determined by RP-HPLC and GP-hPLC.

[0115] After storage at 2-8 ° C for a period of up to three months, the aqueous compositions will show negligible changes in pH. The non-denaturalizing HPLC by size exclusion performed on samples stored for up to three months at 2-8 ° C will not show any significant aggregation of the drug product at pH values of 5.5. The RP-HPLC performed on these samples will not show any significant increase in fragmentation or oxidation of the protein in the pH range of 4.5-5.5.

Example 4

Physical stability of aqueous compositions with various detergents

[0116] Twelve different compositions were prepared. The compositions were:

rFVIIa 0.75 mg / ml NaCl 2.92 mg / ml CaCl2, 2 H2O 1.47 mg / ml Glycylglycine 1.32 mg / ml Detergent / solubilizer x mg / ml pH 5.5

[0117] The concentrations of the detergents / solubilizers evaluated are shown in the table below.

[0118] The compositions were prepared from a liquid bulk solution of rFVIIa. Stock solutions of detergents / solubilizers were prepared in buffers with NaCl, CaCl2, 2 H2O, and glycylglycine at the concentrations shown above. Bulk solutions of rFVIIa and detergent solutions were mixed, and the pH in the solutions was adjusted to 5.5. The compositions were filtered (0.2 μm) and filled in vials (1 ml of solution per vial).

[0119] The appearance of the compositions was determined by visual inspection and the absorbance of the composition at 400 nm was determined. Subsequently, the vials were shaken for 19 hours (800 / min) at room temperature. After completing the stirring, the appearance and absorbance at 400 nm were determined. The results are listed in the table below.

Type of detergent

Conc. (Mg / ml) Appearance  Absorbance (400nm)

Before

 After Before After Increase

None (reference)

- Few part. Very cloudy 0.0085 1,4386 1,4301

Tween® 80

0.1 Very few parts Sure, few part. 0.0044  0.0036 -0,0008

Tween® 20

0.1 Very few parts Sure, few part. 0.0039  0.0101 0.0062

Poloxamer 188

1.0 Very few parts Sure, few part. 0.0063  0.0027 -0.0036

Pluronic® F127

1.0 Very few parts Sure, few part. 0.0000  0.0048 0.0048

Polyethylene glycol 400 Polyethylene glycol 4000

0.1 0.5 Very few parts Few part. Cloudy Very cloudy 0.0076 0.0108 1,5708 1.6624 1.5632 1.6516

Brij® 35

0.1 Very few parts Sure, few part. 0.0028  0.0015 -0.0013

Myrj® 59

0.1 Very few parts Sure, few part. 0.0002  0.1110 0,1108

Myrj® 52

0.1 Very few parts Sure, few part. 0.0009  0.9390 0.9381

LPCM

 0.1 Very few parts Sure, few part. 0.0026  0.0012 -0.0014

Glycerol

 1.0 Very few parts Cloudy, shady 0.0040 1,4064 1,4024

"Part." = "particles"

[0120] The results show that the reference (without the addition of any detergent / solubilizer) becomes visually cloudy when stirred and a significant increase in absorbance is observed at 400 nm. The addition

10 of Tween® 20 (= polysorbate 20), Tween® 80 (= polysorbate 80), poloxamer 188, Pluronic® F127 (= poloxamer 407), Brij® 35 (= polyoxyl 23 lauryl ether), and LPCM (= a-lysophosphatidylcholine myristoyl) almost completely prevented an increase in turbidity and absorbance, while a slight increase in turbidity was observed (compared to the reference) for Myrj® 59 (= polyoxyl 100 stearate) and Myrj® 52 (= polyoxyl 40 stearate) .

15 Example 5

Chemical stability of aqueous compositions containing methionine as an antioxidant

[0121] 20 (I) Three different compositions were prepared. The compositions were:

rFVIIa 0.75 mg / ml NaCl 2.92 mg / ml CaCl2, 2 H2O 1.47 mg / ml Glycylglycine 1.32 mg / ml Methionine 0 or 0.25 or 1.0 mg / ml pH 6.5

The compositions were prepared from a liquid bulk solution of rFVIIa. Methionine was dissolved in buffers with NaCl, CaCl2, 2 H2O, and glycylglycine at the concentrations shown above. The solution to

25 rFVIIa bulk and the methionine solution were mixed, and the pH in the solutions was adjusted to 6.5. The compositions were filtered (0.2 μm) and filled in vials (1 ml of solution per vial). The vials were stored at 5 ° C, 25 ° C and 40 ° C. Samples were removed and analyzed for the content of oxidized forms (by RP-HPLC) at the same time point shown in the table below. The table shows the content of oxidized forms (in%).

Methionine (mg / ml)

Zero time 25rC 14 days 40rC 14 days 25rC 28 days 40rC 28 days 5rC 90 days

0 (reference)

2.4 4.4 7.5 4.4 12.8 3.1

0.25

1.7 2.4 5.3 2.8 9.9 1.9

1.0

1.6 2.3 5.0 2.6 9.6 1.3

The results show that the addition of methionine slows the oxidation rate in the composition.

(II) Three different compositions are prepared. The compositions are:

rFVIIa 0.75 mg / ml NaCl 11.68 mg / ml (200mM) CaCl2, 2 H2O 1.47 mg / ml Glycylglycine 1.32 mg / ml Methionine 0 or 0.25 or 1.0 mg / ml pH 6, 5

The compositions are prepared from a liquid bulk solution of rFVIIa. Methionine is dissolved in buffers with NaCl, CaCl2, 2 H2O, and glycylglycine to obtain the concentrations shown above. The bulk solution of rFVIIa and the methionine solution are mixed, and the pH in the solutions is adjusted to 6.5. The compositions are filtered (0.2 μm) and placed in vials (1 ml of solution per vial). The vials are stored at

15 5 ° C, 25 ° C and 40 ° C. Samples are removed and analyzed for the content of oxidized forms (in%) (by RP-HPLC) at the same time point shown in the table above.

[0122] The results will show that the addition of methionine slows the oxidation rate in the composition.

20 Example 6

Chemical stability of aqueous compositions with sodium chloride

[0123] Four different compositions are prepared. The compositions are:

25 rFVIIa 1.0 mg / ml NaCl 2.92 mg / ml (50mM; composition 1)

11.68 mg / ml (200mM; composition 2) 23.36 mg / ml (400mM; composition 3) 46.42 mg / ml (800mM; composition 4) 58.4 mg / ml (1000mM; composition 5)

CaCl2, 2 H2O 1.47 mg / ml (10mM) Glycylglycine 1.32 mg / ml pH 7.0

[0124] The compositions are prepared from a bulk liquid solution of rFVIIa. The calcium chloride is dissolved in buffers with NaCl and glycylglycine to give the concentrations shown above after mixing with rFVIIa in bulk. After mixing, the pH in the solutions is adjusted to 7.0. The compositions are filtered (0.2 μm) and placed in vials (1 ml of solution per vial). The vials are stored at 5 ° C.

[0125] For each formulation the activity of factor VII (IU / ml) is determined by a coagulation test in 0 months (IU / ml measurement) and after 3 months of storage at 5 ° C (IU / ml measurement). Coagulation assays

35 will show that compositions with at least 200mM sodium chloride (compositions 2-5) have a higher coagulation activity after 3 months of storage (greater IU / ml) compared to composition 1.

Heavy chain degradation

[0126] For each formulation, heavy chain fragment formation is measured by RP-HPLC as described in example 1. Results (as% of fragments) are measured in 0 months as well as during storage at 5 ° C and 30 ° C, respectively; The content of heavy chain fragments (as% of fragments) are measured at 1, 2, 3, and 6 months.

[0127] The measurements will show that the formation of heavy degradation fragments in compositions 2-5 (as% of fragments) is less than the formation of fragments in composition 1.

5 Example 7

Chemical stability of aqueous compositions with sodium chloride

[0128] Four different compositions were prepared. The compositions were:

10 rFVIIa 1.0 mg / ml NaCl 0 mg / ml (0mM; composition 1)

29.2 mg / ml (500mM; composition 2) 43.8 mg / ml (750mM; composition 3) 58.4 mg / ml (1000mM; composition 4)

CaCl2, 2 H2O 1.47 mg / ml (10mM) PIPES-di-Na 17.3 mg / ml (50mM) pH 6.5

[0129] The compositions were prepared from a liquid bulk solution of rFVIIa. The liquid bulk solution was desalted in the desired compositions using Pharmacia PD-10 columns.

[0130] After desalination, the pH in the solutions was adjusted to 6.5. The compositions were filtered (0.22 μm) and filled in cartridges. The cartridges were stored at 5 ° C.

[0131] For each formulation the content of heavy chain fragments (%) was measured by RP-HPLC (example 20 1) at various time points, the data is shown in the table below.

Heavy chain fragment content (%)

Composition

Storage time at 5 ° C (months)

0

QuickTime ™ and aTIFF (uncompressed) decompressoare needed to see this picture. one 2 3

one

11.6 15.6  19.0  24.1  27.8

2

12.2 13.2  15.3  16.7  18.9

3

12.2 13.2  14.6  16.0  18.1

4

12.2 13.0  14.4  15.3  17.1

[0132] It can be seen that increasing the concentration of NaCl reduces the formation of heavy chain fragments.

[0133] For formulations 1 and 4, the coagulation activity of factor VII (IU / ml) was determined at several time points. In the table below the coagulation activity at the various time points has been listed in relation to the zero point of time value (= 100%).

Coagulation activity

Composition

Storage time at 5 ° C (months)

0

3 eleven

one

100% 85% 67% *

4

100% 88% 82%

*: 8 month value

[0134] It can be seen that coagulation activity is higher in composition 4 containing NaCl.

Example 8 Effect of ionic strength on chemical stability [0135] Four formulations were prepared by mixing the bulk solution of purified rFVIIa with solutions

mother containing excipients. In the formulations the content of NaCl and CaCl2 were varied as shown in the table below.

Formulation

mg / mL NaCl mg / mL CaCl2

TO

2.92 1.47

B

58.4 1.47

[0136] In addition, all formulations contained 1. mg / ml rFVIIa, 10 mM glycylglycine, 10 mM sodium acetate, and 10 mM L-histidine. The pH was adjusted to 7.0 in all formulations.

[0137] The formulations were stored at 30 ° C and the content of heavy chain fragments were measured by RP-HPLC (example 1). The results (as% of fragments) are listed in the table below. 10

Formulation

0 months 1 month 2 months 3 months

TO

12.7 31.3 39.1 43.6

B

9.8 19.1 23.2 26.7

[0138] The results show that greater ionic strength (here obtained by the addition of NaCl) produces a decreased formation of heavy chain fragments during storage.

Claims (37)

one.

Liquid aqueous composition comprising a factor VII polypeptide (i); A buffering agent (ii) suitable for maintaining the pH in the range of 5.5 to 9.0; An agent (iii) selected from the list of: a calcium salt, a magnesium salt, or a mixture thereof; where the concentration of (iii) is less than 15 mM; and An ionic strength modifying agent (iv); where the ionic strength of the composition is at least 200 mM.

2.

Composition according to any one of claims 1 to 4, wherein the ionic strength of the composition is at least 250 mM, such as at least 300 mM, 400 mM, 800 mM, 1000 mM, 1200 mM, 1500 mM, 1800 mM, 2000 mM , or at least 2200 mM.

3.

Composition according to claim 1 or claim 2, wherein the ionic strength modifying agent

(iv) is selected from the list of: a neutral salt, for example, sodium chloride; an amino acid; or a small peptide, or a mixture of at least two of said modifying agents.

Four.

Composition according to claim 3, wherein the ionic strength modifying agent (iv) comprises sodium chloride.

5.

Composition according to any one of claims 1 to 4, wherein the agent (iii) is present in a concentration of at least 2 mM, such as at least 5 mM.

6.

Composition according to any one of claims 1 to 5, wherein the calcium salt is selected from the list of: calcium chloride, calcium acetate, calcium gluconate, and calcium levulate.

7.

Composition according to any one of claims 1 to 6, wherein the magnesium salt is selected from the list of: magnesium chloride, magnesium acetate, magnesium sulfate, magnesium gluconate, and magnesium levulate.

8.

Composition according to claim 6 or claim 7, wherein the agent (iii) is selected from the list of: calcium chloride, calcium acetate, magnesium chloride, magnesium acetate, magnesium sulfate, or a mixture thereof; and where the ionic strength modifying agent (iv) is sodium chloride.

9.

Composition according to any one of claims 1 to 8, further comprising a tonicity modifying agent (v).

10.

Composition according to claim 9, wherein the tonicity modifying agent (v) is selected from the list of: a neutral salt; a mono-, di- or polysaccharide; a sugar alcohol; an amino acid; or a small peptide, or a mixture of at least two of said modifying agents.

eleven.

Composition according to claim 9 or claim 10, wherein the tonicity modifying agent

(v) is present in a concentration of 1 mM to 500 mM.

12.

Composition according to claim 11, wherein the concentration of the tonicity modifying agent (iv) is 10-250 mM.

13.

Composition according to any one of claims 1 to 12, further comprising a non-ionic surfactant (vi).

14.

Composition according to claim 13, wherein the non-ionic surfactant is a polysorbate or a poloxamer or a polyoxyethylene alkyl ether such as poloxamer 188, poloxamer 407, polysorbate 20, polysorbate 80, or polyoxy 23 lauryl ether.

fifteen.

Composition according to any one of claims 1 to 14, further comprising an antioxidant (vii).

16.

Composition according to claim 15, wherein the antioxidant (vii) is selected from the list of: L- or Dmethionine, a methionine analogue, a peptide containing methionine, a methionine homologue, ascorbic acid, cysteine, homocysteine, glutathione, cystine , and cystathionine.

17.

Composition according to claim 16, wherein the antioxidant is L-methionine.

18.

Composition according to any of claims 15 to 17, wherein the antioxidant is present in a concentration of 0.1 to 5.0 mg / ml, such as 0.1 to 4 mg / ml, 0.1 to 3 mg / ml, 0.1 to 2 mg / ml, or 0.5 to 2 mg / ml.

19.

Composition according to any one of claims 1 to 18, wherein the pH is maintained in the range of 5.5 to 7.0, or 5.5 to 6.5.

twenty.

Composition according to any one of claims 1 to 19, wherein the agent suitable for maintaining the pH in the range of 5.5 to 7.0 is selected from the list of acids and salts of:

MONTH, PIPES, ACES, BES, TES, HEPES, TRIS, histidine (eg L-histidine), imidazole, glycine, glycylglycine, glycinamide, phosphoric acid (eg sodium or potassium phosphate), acetic acid (eg, ammonium, sodium or calcium acetate), lactic acid, glutaric acid, citric acid (eg sodium or potassium citrate), tartaric acid, malic acid, maleic acid, and succinic acid, or a mixture of at least two of said agents.

twenty-one.

Composition according to claim 20, wherein the concentration of the agent is from 1 mM to 50 mM.

22

Composition according to claim 21, wherein the concentration of the buffer is approximately 10 mM.

2. 3.

Composition according to any one of claims 1 to 22, further comprising a preservative (viii), such as phenol, benzyl alcohol, ortho-cresol, meta-cresol, para-cresol, methyl paraben, propyl paraben, benzalkonium chloride, or chloride Benzethonium

24.

Composition according to any one of claims 1 to 23, which is adapted for parenteral administration.

25.

Composition according to any one of claims 1 to 24, which is stable for at least 6 months at 2-8 ° C.

26.

Composition according to any one of claims 1 to 25, wherein the factor VII polypeptide is human factor VIIa, preferably human factor VIIa obtained by recombination.

27.

Composition according to any one of claims 1 to 25, wherein the factor VII polypeptide is a variant of the factor VII sequence.

28.

Composition according to claim 27, wherein the ratio between the activity of the factor VII polypeptide and the activity of the native human factor VIIa (natural FVIIa) is at least 1.25, preferably at least 2.0 or 4.0, more preferably at minus 8.0, when evaluated in the "In vitro proteolysis assay" as described in the present specification.

29.

Composition according to any one of claims 1 to 28, wherein the factor VII polypeptide is present in a concentration of 0.1 mg / ml to 15 mg / ml, such as 0.5 to 10.0 mg / ml, of 0 , 5 to 5.0 mg / ml, 0.6 to 4.0 mg / ml, or 1.0 mg / ml to 4.0 mg / ml.

30

Method for preparing a liquid aqueous composition of a factor VII polypeptide, comprising the step of providing the factor VII polypeptide in a solution comprising a buffer agent (ii) suitable for maintaining the pH in the range of 5.5 to 9 , 0; an agent (iii) selected from the list of: a calcium salt, a magnesium salt, or a mixture thereof; where the concentration of (iii) is less than 15 mM; and an ionic strength modifying agent (iv); while ensuring that, in the final composition, the ionic strength is at least 200 mM.

31.

Liquid aqueous pharmaceutical composition as defined in any of claims 1-30 for use as a medicament.

32

Use of a composition as defined in any one of claims 1 to 30 for the preparation of a medicament for treating a factor VII sensitive syndrome.

33.

Method for reducing the degradation of a factor VII polypeptide in a liquid formulation, said method comprising the step of providing the factor VII polypeptide in a solution comprising a buffer agent (ii) suitable for maintaining the pH in the range of 5, 5 to 9.0; an agent (iii) selected from the list of: a calcium salt, a magnesium salt, or a mixture thereof; where the concentration of (iii) is less than 15 mM; and an ionic strength modifying agent (iv); while ensuring that, in the final composition, the ionic strength is at least 200 mM.

3. 4.

Method according to claim 33, wherein the concentration of Ca + 2 is at least 2 mM, such as at least 5 mM.

35

Airtight container, at least partially filled, containing an aqueous pharmaceutical composition

liquid as defined in any one of claims 1-31, and optionally an inert gas, said container (i) comprising a wall part and (ii) one or more closing means that do not constitute part of said wall part . 36. Container according to claim 35, wherein the composition does not comprise an antioxidant (vii). 37. A container according to any of claims 35-36, wherein said container is a vial or cartridge comprising a closure means comprising a self-sealed elastomeric partition that can be penetrated by a needle. 38. A container according to claim 37, wherein said container is a cartridge further comprising a movable piston means through which the liquid present in said container can be ejected from said container.